Pathogens express their virulence genes in response to specific signals detected during infection. One critical signal is mildly acidic pH, which is critical to induce virulence genes and to promote tolerance to multiple antibiotics in the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. In addition, a mildly acidic pH activates four different virulence regulatory systems: the ancestral PhoP/PhoQ, PmrA/PmrB and OmpR/EnvZ, and the horizontally acquired SsrB/SpiR. We have now determined that activation of the PhoP/PhoQ system by mildly acidic pH is dependent on the regulatory gene ssrB; and that contrary to the prevailing model, Salmonella responds to mildly acidic pH even when the senor PhoQ lacks its extracytoplasmic domain. This proposal describes experiments to determine the mechanism by which SsrB enables the PhoP/PhoQ system to respond to mildly acidic pH; to define the PhoQ residues and the precise nature of the cytoplasmic signal acting on PhoQ when Salmonella experiences a mildly acidic pH; to identify the PhoP-regulated genes responsible for acid resistance and to examine their virulence role; and to examine the activities of the PhoP/PhoQ, PmrA/PmrB, OmpR/EnvZ and SsrB/SpiR when Salmonella is inside different types of macrophages and in response to mildly acidic pHs. The proposed research will establish new paradigms in acidic pH sensing and in the understanding of the evolution of bacterial pathogens.